The present invention relates to scanning microscopes. More particularly, the present invention relates to a scanning microscope which allows morphological observation and fluorescence observation to be performed simultaneously and enables both a morphological observation image and a fluorescence observation image to be obtained within a reduced period of time.
A technique called xe2x80x9clow-coherence interferometryxe2x80x9d such as that disclosed in U.S. Pat. No. 5,321,501 is known as a method that allows observation of the inside of an opaque scattering sample, e.g. a biological tissue. FIG. 11 shows a typical optical system for the low-coherence interferometry. Light from a light source 81 with a short coherence length is split by a beam splitter 82 between a signal light path leading to a sample 5 and a reference light path leading to a reflecting mirror 83. Light going and returning along the signal light path and the reference light path are recombined in the beam splitter 82. At this time, because the signal light path forms an optical path length substantially equal to that of the reference light path at an observation position 6 within the sample 5, only light scattered back from a region at the observation position 6 within a range in the optical axis direction that is substantially equal to the coherence length interferes with the reference light. Accordingly, by detecting the resulting interference signal with a detector 84, information about the inside of the sample 5 can be selectively obtained in the optical axis direction. In general, the reflecting mirror 83 in the reference light path is moved in the optical axis direction, thereby performing scanning in the direction of depth of the sample and, at the same time, giving a Doppler shift to the reference light. The low-coherence interferometry generally includes heterodyne interferometric measurement that is carried out to detect a beat signal having a Doppler shift frequency in the interference signal. Therefore, the measurement can be performed with a very high S/N ratio. Accordingly, if near infrared light or the like is used as the light source 81, it is possible to detect feeble scattered light from a position as deep as several millimeters from the surface of the scattering sample 5. By scanning the signal light or the sample 5 in a plane perpendicular to the optical axis, it is possible to obtain an image of a section perpendicular to the optical axis.
Meanwhile, a low-coherence interferometric method is published in xe2x80x9cJournal of Modern Opticsxe2x80x9d, Vol. 45, No. 4, p.765 (1998), in which acoustooptic devices are disposed in the signal light path and the reference light path, respectively, and a beat signal corresponding to the difference between the modulation frequencies of the acoustooptic devices is detected without moving the reflecting mirror.
Incidentally, a fluorescence observation method is known as an observation method for biological samples or the like. According to the fluorescence observation method, a cellular tissue or a specific substance is labeled with a fluorescent dye, and a fluorescence image produced when excitation light is applied to the sample is observed. The sample may be sliced for microscopic observation. Recently, however, there have been increasing needs to observe biological samples or the like in a living state, and there has been a growing demand for obtaining a fluorescence image at some depths from the sample surface.
During fluorescence observation, it is desirable to be possible to simultaneously obtain morphological information about a fluorescence-labeled tissue or substance and information concerning a surrounding spatial structure. However, it is difficult to obtain information about the inside of a thick sample in ordinary microscopic observation. In observation of such morphological information, for example, when it is intended to observe changes of biological activities of a living biological tissue with time, it is desirable that the time required for the observation should be as short as possible. When morphological observation or the like is performed simultaneously with fluorescence observation, if excitation light is continuously applied to the fluorescence-labeled sample for a long period of time, the fluorescent dye fades. Consequently, the fluorescence image becomes dark as time goes by. Therefore, in this case also, the time required for observation should be minimized.
The above-described patent and literature give no description of a fluorescence observation method and do not mention an arrangement in which low-coherence interferometric measurement is carried out during fluorescence observation. Such an arrangement is disclosed in U.S. patent application Ser. No. 09/172,676 and Japanese Patent Application Unexamined Publication (KOKAI) No. 11-119106, which were filed by the present applicant. However, the disclosed arrangement uses a method in which the reflecting mirror in the reference light path is moved to perform observation. Therefore, it is necessary to move the reflecting mirror also when observing a section within the sample that is perpendicular to the optical axis. Accordingly, when low-coherence interferometric measurement is carried out simultaneously with fluorescence microscopic observation, in which, generally, a section perpendicular to the optical axis is observed, the time required for measurement undesirably lengthens by an amount corresponding to the time needed for mechanical drive of the reflecting mirror.
In view of the above-described problems with the prior art, an object of the present invention is to provide a scanning microscope in which when performing fluorescence observation of the inside of a thick sample or the inside of an opaque scattering sample, it is possible to simultaneously perform morphological observation for obtaining morphological information or the like in the same region of interest as that for the fluorescence observation, and it is possible to obtain both a fluorescence observation image and a morphological observation image within a reduced period of time.
To attain the above-described object, the present invention provides a scanning microscope including a low-coherence light source and a device for splitting low-coherence light from the low-coherence light source between a first optical path and a second optical path. A frequency modulator is placed in at least one of the first and second optical paths to produce a frequency difference between light passing through the first optical path and light passing through the second optical path without changing the optical path length of each optical path. An objective optical system is placed in the first optical path to apply light to a sample and to collect light from the sample. A scanning device is placed in the first optical path to scan the sample and the light applied by the objective optical system relative to each other in a plane perpendicular to the optical axis of the objective optical system. The scanning microscope further includes a device for combining together the first and second optical paths, and an interference signal detecting system for detecting an interference signal having the frequency difference from the combined light. A fluorescence branching device branches fluorescence from the sample excited by the low-coherence light. A fluorescence detecting system detects the fluorescence branched by the fluorescence branching device.
The arrangement and operation of the scanning microscope according to the present invention will be described below with reference to FIG. 1, which shows the arrangement of the scanning microscope according to the present invention.
Low-coherence light from a low-coherence light source 1 is split between a first optical path and a second optical path by an optical path splitting device 2. In FIG. 1, an optical path along which light reflected by the optical path splitting device 2 travels is defined as a first optical path, and an optical path along which light passing through the optical path splitting device 2 travels is defined as a second optical path. Frequency modulators 3a and 3b are placed in the respective optical paths. The frequency modulators 3a and 3b have been set to produce a frequency difference f between light passing through the first optical path and light passing through the second optical path without changing the optical path length of each optical path. The arrangement may be such that a frequency modulator is provided in only either one of the first and second optical paths. The light passing through the first optical path is applied to a sample 5 through an objective optical system 4. Light reflected and scattered by the sample 5 passes through the objective optical system 4 again. Then, the light is combined with the light passing through the second optical path by devices 7 and 8 for combining the first and second optical paths and led to an interference signal detecting system 9. The interference signal detecting system 9 extracts a beat signal of frequency f from the detected signal. The amplitude of the beat signal is determined to be scattered light intensity information. In the light scattered from the inside of the sample 5, light from the vicinity of an observation position 6 in the sample 5, at which the first optical path has an optical path length substantially equal to that of the second optical path, interferes with the light passing through the second optical path. The above-described vicinity of the observation position 6 is within a range in the optical axis direction that is substantially equal to the coherence length of the low-coherence light source 1. Accordingly, scattered light intensity information in the sample 5 can be obtained with z-resolution substantially equal to the coherence length.
If a fluorescence label excitable by light from the low-coherence light source 1 has been introduced into the sample 5, it is possible to perform fluorescence observation of a specific tissue, substance, etc. in the sample 5. It is not necessary to introduce a fluorescence label when a tissue of interest fluoresces by itself to the wavelength of light from the low-coherence light source 1. Fluorescence emitted from the sample 5 is separated from the low-coherence light by a fluorescence branching device 10 and led to a fluorescence detecting system 11.
Accordingly, the same region 6 in the sample 5 can be observed simultaneously by both the low-coherence interferometric observation method and the fluorescence observation method. In addition, an image of a section in the sample 5 that is perpendicular to the optical axis can be obtained by scanning the light applied to the sample 5 by the objective optical system 4 in a plane (xy-section in FIG. 1) perpendicular to the optical axis of the objective optical system 4 by using a scanning device 12. The sample 5 may be scanned instead of scanning the light applied to the sample 5 by the objective optical system 4.
In the arrangement shown in FIG. 11, even when one point in an xy-section of the sample 5 is observed, the optical path length of the reference light path is changed to produce a frequency difference between the reference light path and the signal light path while scanning the sample 5 in the z-direction. The frequency modulators 3a and 3b in the present invention do not change the optical path lengths of the first and second optical paths. Therefore, observation of one point in an xy-section needs no mechanical drive. In the scanning microscope according to the present invention, there is always a frequency difference f between the first optical path and the second optical path. Accordingly, it is possible to obtain a low-coherence interferometric image of an xy-section at a much higher speed than in the case of the arrangement shown in FIG. 11.
The low-coherence light source 1 may be a pulse laser. In this case, the fluorescence dye can be subjected to multiphoton excitation by the pulse laser. In multiphoton excitation, only the fluorescence dye near the condensed excitation light spot is excited, and no fluorescence is emitted from a region away from the observation plane even if it is irradiated with the excitation light. Therefore, excess background light is suppressed, and the S/N ratio of fluorescence observation is increased.
The fluorescence branching device 10 may be a dichroic mirror. Optimally setting the reflection-transmission characteristics of the dichroic mirror allows efficient separation of fluorescence and scattered light from the sample 5 and thus makes it possible to improve the efficiency of detection of fluorescence and scattered light.
In addition, the present invention provides another scanning microscope including a low-coherence light source and a device for splitting low-coherence light from the low-coherence light source between a first optical path and a second optical path. A frequency modulator is placed in at least one of the first and second optical paths to produce a frequency difference between light passing through the first optical path and light passing through the second optical path without changing the optical path length of each optical path. An objective optical system is placed in the first optical path to apply light to a sample and to collect light from the sample. A scanning device is placed in the first optical path to scan the sample and the light applied by the objective optical system relative to each other in a plane perpendicular to the optical axis of the objective optical system. The scanning microscope further includes a device for combining together the first and second optical paths, and an interference signal detecting system for detecting an interference signal having the frequency difference from the combined light. Further, the scanning microscope includes a fluorescence excitation light source, and an excitation light combining device for combining together excitation light from the fluorescence excitation light source and the low-coherence light in the first optical path. A fluorescence branching device branches fluorescence from the sample excited by the excitation light. A fluorescence detecting system detects the fluorescence branched by the fluorescence branching device.
The arrangement and operation of the second scanning microscope according to the present invention will be described below with reference to FIGS. 2 and 3, which show the arrangement of the second scanning microscope according to the present invention.
The second scanning microscope is similar to the scanning microscope shown in FIG. 1 in the arrangement of a part in which light from a low-coherence light source 1 is split between two optical paths by an optical path splitting device 2, and a beat signal having a frequency difference produced by frequency modulators 3a and 3b is detected by an interference signal detecting system 9. Therefore, a description of this part is omitted. The arrangement shown in FIG. 2 differs from the arrangement shown in FIG. 1 in that the scanning microscope has a fluorescence excitation light source 15 in addition to the low-coherence light source 1. In FIG. 2, excitation light from the fluorescence excitation light source 15 is combined with low-coherence light in the first optical path by an excitation light combining device 16 and applied to a sample 5, together with the low-coherence light. Fluorescence emitted from the sample 5 is separated by a fluorescence branching device 17 and led to a fluorescence detecting system 11. The excitation light combining device and the fluorescence branching device may be arranged to perform each other""s function as well as their own functions, as shown by reference numerals 18 and 19 in FIG. 3. That is, in FIG. 3, both the devices 18 and 19 are excitation light combining devices and also fluorescence branching devices.
In the present invention, the scanning microscope has the fluorescence excitation light source 15 separately from the low-coherence light source 1. Therefore, it is possible to select light sources of wavelengths that are most suitable for low-coherence interferometric observation and fluorescence observation, respectively.
The second scanning microscope according to the present invention also allows the same region 6 in the sample 5 to be observed simultaneously by both the low-coherence interferometric observation method and the fluorescence observation method as in the case of the scanning microscope shown in FIG. 1. In addition, an image of an xy-section can be obtained by the scanning device 12, and thus high-speed two-dimensional image observation can be performed.
The excitation light combining device and the fluorescence branching device can be formed from at least two dichroic mirrors. Thus, low-coherence light and fluorescence can be separated from each other efficiently.
The arrangement may be such that one of the excitation light combining device and the fluorescence branching device is a dichroic mirror, and the other is a mirror capable of switching between optical paths. For example, the scanning microscope shown in FIG. 2 may be arranged such that the device denoted by reference numeral 16 is a mirror capable of switching between optical paths, and the device denoted by reference numeral 17 is a dichroic mirror. When, as shown in FIG. 2, the device 16 is placed as a mirror in the first optical path, only fluorescence observation is performed. When both the devices 16 and 17 are withdrawn from the first optical path, only low-coherence interferometric observation is performed. With this arrangement, it is possible to switch between fluorescence observation and low-coherence interferometric observation. This is useful when scattered light and fluorescence from the sample 5 are feeble and it is therefore desired to minimize the loss of light quantity. In FIG. 3, the same action can be obtained by using a mirror capable of switching between optical paths as the device denoted by reference numeral 19 and further using a dichroic mirror as the device denoted by reference numeral 18.
The frequency modulators used in the above-described scanning microscopes according to the present invention may be acoustooptic devices. Acoustooptic devices can readily produce frequency modulation in accordance with the driving frequency thereof and are therefore suitable for the heterodyne measurement in low-coherence interferometry.
In the foregoing scanning microscopes according to the present invention, at least one of the interference signal detecting system and the fluorescence detecting system may be arranged in the form of a confocal system. For example, pinholes or the like are placed on the illumination side and immediately in front of each detecting system, respectively, to form a confocal detecting system. Thus, it is possible to increase the spatial resolution in either or both of low-coherence interferometric observation and fluorescence observation.
In the foregoing scanning microscopes according to the present invention, at least one of the first and second optical paths may be provided with at least one dispersion adjusting device for substantially equalizing dispersion characteristics produced in the first and second optical paths, respectively, in the wavelength region of the above-described low-coherence light. Because low-coherence light has a spectral width of certain size, when it passes through each optical device provided in the optical paths, the optical path length of the light differs for different wavelengths according to the dispersion characteristics of each optical device. For example, if the spectral distribution of the low-coherence light source 1 is as shown in FIG. 12, when light from the low-coherence light source 1 passes through an optical device having dispersion characteristics as shown in FIG. 13, the optical path length of light of xcexl shown in FIG. 13 becomes longer than the optical path length of light of xcexh. In the scanning microscope according to the present invention, low-coherence light split between the first optical path and the second optical path is affected by the dispersion of each optical device in each optical path before being recombined. If the first and second optical paths are different in dispersion characteristics from each other, the difference in optical path length between the first and second optical paths undesirably varies according to wavelengths. Even if the same optical devices are used in the first and second optical paths, when the sample 5 itself has dispersion, the difference in optical path length between the first optical path, which passes through a part of the sample 5, and the second optical path, which does not pass through any part of the sample 5, varies according to wavelengths, undesirably. If the optical path length difference varies according to wavelengths, the observation position 6 in the sample 5 where an interference signal is detected in FIG. 1, for example, varies in the z-axis direction according to wavelengths. Consequently, the resolution and the S/N ratio degrade unavoidably.
Therefore, in the scanning microscopes according to the present invention, at least one of the first and second optical paths is provided with at least one dispersion adjusting device for substantially equalizing dispersion characteristics produced in the first and second optical paths, respectively, in the wavelength region of the low-coherence light, thereby solving the above-described problem.
The at least one dispersion adjusting device provided in the second optical path may be variable in optical thickness. For example, to observe images of sections (in the xy-plane) perpendicular to the optical axis successively in the direction of depth of the sample in order to obtain information concerning a three-dimensional structure inside the sample 5, after each observation of an image in the xy-plane, either the objective optical system 4 or the sample 5 is moved in the z-direction. At this time, the optical path length of the first optical path changes. Therefore, it is necessary to change the optical path length of the second optical path in accordance with the change in optical path length of the first optical path. Because the change in optical path length of the first optical path includes a change in optical path length of light passing through a part of the sample 5, if the sample 5 has dispersion, the influence of dispersion in the first optical path changes correspondingly. Accordingly, simply changing the air spacing between the optical devices in the second optical path with respect to the change in optical path length of the first optical path allows the optical path lengths of the first and second optical paths to become equal to each other but cannot equalize the influence of dispersion in the first and second optical paths with each other. The above-described arrangement makes it possible to change the optical thickness of the at least one dispersion adjusting device placed in the second optical path. Therefore, it is possible to adjust the dispersion in accordance with a change in dispersion influence caused by the sample 5, which results from a change in z-coordinate of the observation plane or the like. When there has been a change in influence of the dispersion in the microscope optical path, e.g. when the sample 5 has been changed for another, or when an optical device having dispersion has been newly placed in the microscope optical path, it is also possible to perform an optimal dispersion adjustment at the time of carrying out observation.
The scanning microscope may be arranged such that he depth of the observation plane in the sample 5 from the surface of the sample 5 can be adjusted by changing the optical thickness of the dispersion adjusting device. Changing the optical thickness of the dispersion adjusting device causes a change in optical path length of the second optical path and hence causes a change in z-coordinate of the observation position 6 in the sample 5, at which the first optical path has an optical path length substantially equal to that of the second optical path. If the depth of focus of the objective optical system 4 is sufficiently greater than the coherence length, the z-coordinate of the observation position 6 can be changed without substantially changing the resolution in the xy-plane. Accordingly, the depth (z-coordinate) of the observation plane in the sample 5 can be changed by changing the optical thickness of the dispersion adjusting device. As has been stated above, the dispersion adjusting device makes it possible to perform an optimal dispersion adjustment at the time of carrying out observation. Therefore, it is possible to simultaneously adjust the depth of the observation plane in the sample 5 and the dispersion, which may be changed by the depth adjustment.
The scanning microscope may be arranged so that the dispersion characteristics of the dispersion adjusting device in the wavelength region of the low-coherence light are substantially equal to the dispersion characteristics of the sample 5. If the optical thickness of the dispersion adjusting device is set at a value approximately double the depth of the observation position 6 in the sample 5 from the surface of the sample 5, the optical path length of light passing through the dispersion adjusting device and the optical path length of light passing through a part of the sample 5 become substantially equal to each other. Therefore, it is possible to substantially equalize the influence of dispersion introduced by the sample 5 and the influence of dispersion introduced by the dispersion adjusting device. Accordingly, it is possible to further facilitate the above-described simultaneous adjustment, that is, the adjustment of the depth of the observation plane and the adjustment of the dispersion, which may be changed by the depth adjustment.
Still other objects and advantages of the invention will in part be obvious and will in part be apparent from the specification.
The invention accordingly comprises the features of construction, combinations of elements, and arrangement of parts which will be exemplified in the construction hereinafter set forth, and the scope of the invention will be indicated in the claims.